Detection of heat stable proteins from meat of ruminant

ABSTRACT

Disclosed is a method for pretreating heat stable proteins derived from a rendered animal material prior to performing an assay for their presence comprising (a) preparing a protein containing extract of the material, (b) removing substantially all or part of the gelatin from the extract and (c) concentrating the remaining protein such that it tests positive for protein by immunoassay at a dilution of greater than 1 in 6,000.

This application is a 371 of PCT/GB94/01715 filed Aug. 4, 1994.

The present invention relates to a method for the detection of proteinsin rendered animal material, and particularly a method for detectingheat stable ruminant proteins in such material.

A ban on rendered ruminant protein in ruminant feedstuffs was institutedby the United Kingdom Ministry of Agriculture, Fisheries and Food inJuly 1988 in response to outbreak of Bovine SpongiformEncephalopathy--the Bovine Spongiform Encephalopathy Order 1988(Statutory Instrument 1988/1039). This, and the serious nature of thisdisease, provides a need to detect these proteins in feedstuffs.

Animal material that passes through a rendering process is subjected totemperatures ranging from 102° C. to 145° C. with a typical exposuretime of up to two hours. Current immunological tests aimed atdetermining the species of origin of resulting meat and bonemeal havelimited capability, with little or no sensitivity at below 1%contamination when speciating material cooked above 130° C. for morethan one and a half hours; these temperatures being confirmed bydifferential scanning calorimetry.

Meat speciation is commonly carried out using ELISA systems based onantibodies against muscle heat stable proteins. Berger et al, (1988) J.Association of Official Analytical Chemists, 7, (2), 406-409, have usedantisera raised to raw skeletal muscle to detect pork and chicken heatedto 120° C. for 15 minutes, while Kang'ethe and Gathuma (1987) MeatScience 19, 265-270, used antisera against cooked muscle proteins,mainly from ruminants, with similar effect. Heat stable proteins havealso been demonstrated as present in adrenal glands, brain, testes,heart and kidney.

Material for rendering is composed mainly of bone, fat and viscera, withskeletal muscle being a lesser component. Collagen, which is present inall tissues, converts to a soluble form when heated to become the mainconstituent of gelatin. Water soluble extracts of meat and bonemealfrequently solidify due to the high gelatin content and this gelatin iscapable of depressing the immunoassay sensitivity (e.g. see results inKang'ethe and Linqvist (1987) in Journal of Science of Food &Agriculture, 39, 179-184.

Thus the immunoassay of heat stable protein in rendered materials suchas meat and bonemeal is problematical. Commercial kits currentlyavailable can detect bovine and ovine species in these materials at adilution no greater than one part in 100, and this level of sensitivitydecreases in materials that have been treated under high temperaturerendering conditions, ie. at about 135° C.-155° C. for up to one and ahalf hours.

The heat stable sample preparation regimes of Berger et al and Kang'etheand others can be summarised as follows:

(i) The method of Berger et al (1988) mixes cooked or canned meat withdistilled water over a short periods e.g. 1 minutes leaves the mixtureto stand for about one hour at room temperature then centrifuges it, thesupernatant being used for the assay step.

(ii) The method of Kang'ethe et al (1986) Journal of Science of Food &Agriculture, 37, 157-164 homogenises the meat products in saline in aratio of about 1:1 (w/v) and then sonicates the mixture at 300 W for 10minutes, the sonicate is centrifuged at about 2000 g for 15 minutes andresultant supernatant dialysed against saline for 3 days before use inthe assay step.

(iii) The method of Kang'ethe and Lindqvist (1987) is initially similarto that of Kang'ethe et al (1986) but uses fresh meat, and after thecentrifugation step the supernatant is filtered, centrifuged at 86000×gfor 30 minutes, and the resulting supernatant autoclaved at 121° C. for30 minutes. The autoclaved material is again centrifuged at 2000 g for15 minutes and the antigens precipitated from the supernatant byaddition of 3 vols absolute ethanol; the mixture being left overnightand the precipitate being recovered by centrifugation. The precipitateis then dissolved in saline, concentrated by ultrafiltration (30 kDacut-off) and fractionated by gel filtration (Sephadex G-200 or G-75).Fractions containing antigen are concentrated by ultrafiltration forassay.

The present inventor has now provided a method for concentrating heatstable proteins in such materials, thus increasing the sensitivity oftests such as the aforesaid immunoassays when they incorporate suchpreparative method as a preliminary step. This method is surprisinglycapable of detecting low concentrations of heat stable proteins in ovinemuscle and tissue that has been heated to 145° C., and bovine muscle andtissue that has been heated to 155° C., for periods of 90 minutes suchas are found in commercial rendering process.

In a first aspect the present invention provides a method forpretreating heat stable proteins derived from a rendered animal materialprior to performing an assay for their presence comprising (a) preparinga protein containing extract of the material, (b) removing substantiallyall or part of the gelatin from the extract and (c) concentrating theremaining protein, preferably such that it tests positive for protein byimmunoassay at a dilution of 1 in 6,000 for rendered meat and bonemealand 1 in 400 for compound feeds. The present method particularlycomprises preparing a liquid buffered extract of the rendered material,removing solid material, e.g. by filtering, adding a salt to the extractat such a concentration as to cause preferential precipitation ofgelatin, preferably together with other non-specific proteins and oils,e.g. of plant origin, separating this gelatin precipitate from theextract, increasing the concentration of the salt until a substantialportion of the remainder of the protein is precipitated, and isolatingthat protein.

Preferred salts are ammonium salts, particularly ammonium sulphate, butother suitable salts will occur to those skilled in the art. Usingammonium sulphate the preferential precipitation of gelatin isconveniently carried out by adding sufficient of the salt to thebuffered extract to provide between 30 and 70%, preferably between 30and 40%, weight salt solution. In order to precipitate the heat stableproteins once the gelatin has been removed further ammonium sulphate isadded to give a final concentration in excess of 80% weight, morepreferably over 90%, and preferably about 92.5% weight.

The steps of gelatin and heat stable protein removal are convenientlycarried out using centrifugation. Most preferably the extract and saltmix is left to stand for a period of hours, e.g. 6 to 18 hours,preferably 12 hours, before centrifugation, preferably standing at atemperature form 25 to 35° C., most preferably about 30° C. After thisperiod the extract salt mix is centrifuged at between 8000 and 10000 g,preferably about 9000 g, and then is preferably filtered to removecloudy precipitate.

Filtration is conveniently carried out using a micropore filter, e.g. ofabout 1μ, e.g. a 0.6μ filter. The heat stable proteins which remain inthe extract supernatant are isolated preferably by addition of stillmore salt with constant mixing until they precipitate. With ammoniumsulphate this is typically found to occur when the salt saturation is92.5% and the temperature is maintained at about 30° C. as before.Preferably the mix is shaken to ensure that the salt dissolves and theresultant protein precipitate is separated out by centrifugation underconditions used to isolate the gelatin, plant proteins and oils. Oncethe heat stable proteins have been precipitated in this way and washedthey are conveniently resuspended for the immunoassay or other testmethods, whereby the concentration effected is some 30 fold that of theoriginal, with the added advantage that immunological blocking caused bygelatin is avoided.

The buffer used to prepare the initial mix of meat and bonemeal isconveniently such that neutral pH is provided, e.g. pH7.2, and themixture is preferably blended using an homogenizer and filtered.

In a further aspect the present invention provides a method for assayinga rendered animal material for presence, identity and/or amount of heatstable proteins comprising the pretreatment method as described in thefirst aspect of the invention, followed by performance of an immunoassaydirected at one or more of the proteins and relating the presence,identity and/or concentration of such proteins in the extract to theirpresences identity and/or concentration in the rendered animal material.Preferred immunoassays use a binding assays e.g. an ELISA or RIA,directed at a number of such proteins such that rapid screening forpresence of a number of sources is possible in one assay.

In a still further assay of the present invention there is provided atest kit for assaying the presence, identity and/or amount of a heatstable protein present in or derived from a rendered animal material,comprising a salt, in solid or liquid form, suitable for adding to aextract made from a rendered animal material such that gelatin presenttherein will be selectively precipitated while leaving substantially allthe heat stable protein in solution; together with one or more specificbinding agents necessary for carrying out an immunoassay for detectingthe presence, identity or concentration of extract protein.

The kit may omit the salt if preferred assay items are included. Typicalbinding reagents will be specific antibodies raised to heat stableproteins of interest, or to numbers of such proteins. Polyclonal ormonoclonal antibodies, or mixtures of these may be used. Capture assaycomponents, such as immobilised antibodies of a first specificitytogether with solutions of free antibodies to a subset of the firstspecificity, may be provided. In preferred assays the protein fractionprepared by the method of the invention is resuspended in 2% ovalbuminsolution; the antibodies are preferably raised heterologously to bothfresh and cooked meat and tissue and PVP is incorporated into the platewashing solution.

The method of the present invention will now be exemplified by way ofillustration only by reference to the Figures and Examples. Otherembodiments falling into the scope of the claims will occur to thoseskilled in the art in the light of this and the description above.

FIGURES

FIG. 1: shows a graph of protein concentration of supernatant in mg/100ml against percentage ammonium sulphate saturation for solutionscontaining 0.5, 1.0, 1.5 and 2.0% gelatin and indicates the consistencyof the supernatant so produced.

FIG. 2: shows a graph of Optical Density against dilution of extractobtained using the assay of the example for ovine and bovine meat andbonemeals when added to avian/porcine meat and bonemeal.

FIG. 3: shows a graph of the effect of temperature on bovine and ovinetissues at 3 dilutions; 1/1000, 1/3200 and 1/6400; temperatures beingmaintained for 1.5 hours.

FIG. 4a: shows a histogram of OD at 450 nm against dilution of extractswithout blocking agents being present using the assay of the example.

FIG. 4b: shows a histogram of OD at 450 nm against dilution of extractswith PVP and ovalbumin blocking agents present using the assay of theexample.

EXAMPLE

Preparation of antibodies to heat stable proteins for use in method:

Four groups of antigens were produced from each of bovine, ovine andporcine material and two groups from poultry (chicken/turkeymixture)-see Table 1. The bovine and ovine antigens were used asimmunogens and all antigens were used at a later stage to assessantibody sensitivity and specificity.

                  TABLE 1    ______________________________________    Bovine, ovine, porcine and avian antigen groups    ANTIGEN MATERIAL                   BOVINE   OVINE   PORCINE                                           AVIAN    ______________________________________    i  Fresh skeletal muscle                   +        +       +      +    ii   Fresh tissue cocktail                   +        +       +      -    iii  Cooked skeletal muscle                   +        +       +      +    iv  Cooked tissue cocktail                   +        +       +      -    ______________________________________

Bovine and ovine materials were used as immunogens; + indicates used forantigen production; - indicates not used for antigen production.

Fresh muscle antigen: to avoid possible variations due to breed, age andsex, 50×60 g of lean skeletal muscle samples were taken at random fromlocal abattoirs. 20 g was removed from each sample, pooled and theresultant 1 kg minced and divided into four aliquots of 250 g each.Based upon the method of Berger et al (1988), each aliquot was treatedseparately to remove non-essential material and the resulting 4 harvestspooled. An XK50/20 column (Pharmacia) was packed with S Sepharose (TM)Fast Flow Cation resin (Pharmacia) and equilibrated with 0.01 M sodiumacetate starting buffer pH3.7. The system was linked to an automaticgradient system (Pharmacia) and the pooled extract eluted with a lineargradient composed of 180 ml starting buffer and 167 ml of 0.1 M sodiumacetate limit buffer at 20 ml/hour. 3 ml fractions were collected andthe eluant constantly monitored at 280 nm absorbance; excess limitbuffer was added to ensure complete elution of the major peak after thegradient had run out. The fractions containing the major peak weredialysed against 30% polyethyleneglycol (PEG 20,000 Daltons) toapproximately 2 mg protein/ml determined by the Sigma microdetermination method.

The total antigen yield was recorded and the isoelectric point (pI) ofeach of the main proteins was determined by isoelectric focusing usingthe Phast System (TM, Pharmacia UK Ltd).

Fresh tissue antigen: Heart, adrenals, brain, liver, spleen, kidney,lung and testes were taken from 50 randomly selected carcases toovercome variations as described for collection of skeletal muscle.Tissues associated with alimentary tract were deliberately avoidedbecause of the possible presence of ingested plant or animal proteins.Approximately 20 g of material free from connective tissue and fat wereremoved from each tissue and the resulting portions pooled and minced. 1Kg of the pooled cocktail was then used for antigen production andtreated as for fresh muscle with total antigen yield and pI bandlocations recorded.

Cooked skeletal muscle: From each of the original 50×60 g samples oflean skeletal muscle a further 20 g was taken and finely minced.Partially purified thermostable muscle antigen was produced by applyingthe method of Kang'ethe and Gathuma (1987) Meat Science 19, 265-270.Antigen yield and the main pI positions were recorded.

Cooked tissue antigen: A further 1 Kg of the cocktail of fresh tissueswas minced and used for cooked procedure. The method of Kang'ethe andGathuma was again applied as per the cooked skeletal muscle. Antigenyield and the main pI positions were recorded.

Antisera production: Fifteen sandy lop eared rabbits of approximately 3Kg each were immunised using the same inoculation procedure as for both"fresh" and "cooked" immunogens. Prevention of ulceration from theprimary injection was achieved using a non-ulcerative Freund's completeadjuvant (Morris). 0.05 ml of reconstituted BCG vaccine (Glaxo,Intradermal) was added to the aqueous phase for each 2 ml of oilyadjuvant. Rabbits were inoculated subcutaneously at multiple sites ontheir backs with 1 mg of the aqueous immunogen in an equal volume of thecomplete adjuvant. Booster injections were given subcutaneously atmultiple sites at 4-week intervals (Hardlow and Lane (1988) LaboratoryManual CSH p114-116, using 1 mg of the aqueous immunogen in 2 volumes ofoily adjuvant. This continued until a high level of antibody productionwas obtained; antibody titre to the homologous immunogen was checkedprior to and during the inoculation procedure using Ouchterlony'smicrotechnique double diffusion test of Crowle (1973) Immuno Diffusion,2nd ed. pg 286-294; Academic Press, N.Y. Rabbits were exsanguinated whena satisfactory titre was achieved and isolation of antibody (IgG/IgA)was carried out by the method of Axelsen et al (1973). The major proteinfractions, detected using the Sigma micro-detection method for totalprotein, pooled and dialysed against 0.1 M phosphate buffered saline,pH7.0. The protein content was again measured and then the pooledfractions split into 1 ml aliquots and stored at -70° C.

Preparation of meat and bone meal sample extracts and test controls:

Each meat and bonemeal used contained a single species (bovine, ovine,porcine and avian) and was produced by a commercial rendering process at130° C. Each was subjected, in the laboratory, to additional heating to130° C. for 30 minutes, cooled and stored at -20° C. in 40 g amounts.Extracts of protein for immunoassay were made up by applying either themethods of Berger et al (1988), Kang'ethe and Linqvist (1987), Kang'etheet al (1986) with the method of the present invention involving removalof gelatin and concentration.

EXAMPLE 1 Pretreatment Step of the Invention

Meat and bonemeal (10 g) and 0.1 M phosphate buffer pH7.2 (90 ml) weremixed, allowed to stand at room temperature for 15 minutes, homogenisedin a blender (Waring) at 22° C. for 2 minutes, before being filteredthrough 4 layers of muslin and then centrifuged at 8890×g for 30 minutesat 22° C. 20 ml aliquots of filtrate were collected and those notimmediately required stored at -70° C.

A 35% concentration of anhydrous ammonium sulphate was used toprecipitate the gelatins as predetermined using 0.5%, 1.0%, 1.5% and 2%solutions of purified animal gelatins. To achieve this 4.18 g ofanhydrous ammonium sulphate was slowly added with constant mixing toeach aliquot and the mixture allowed to stand for 12 hours at 30° C.,centrifuged at 8890 g for 30 minutes, and any cloudiness removed byfiltering through a 0.6μ filter (Millipore Limited, Watford, UK).Remaining protein was concentrated by addition of 8.68 g of anhydrousammonium sulphate with constant mixing to bring the total ammoniumsulphate saturation to 92.5%. The mixture was kept at 30° C. for 12hours with periodic shaking to ensure all the ammonium sulphatedissolved and the resultant precipitate was separated out bycentrifugation at 8890×g at 22° C. for 30 minutes with the supernatantbeing discarded.

To prevent ammonium sulphate affecting the immunoassay any crystalsremaining were immediately redissolved by the careful addition of 20 mlof deionised water and immediately removed by inversion of the tube. Thewashing was repeated once and the centrifuge tube inverted ontoabsorbent paper and allowed to drain for 30 minutes. The washingprocess, if carried out carefully and quickly, has minimal effect on theprotein precipitate, but removes all traces of the salt. The precipitatewas initially dissolved in 5 drops of 2% Ovalbumin in 0.01 M phosphatebuffered saline and the resulting concentrated protein solution diluted1/5 in 2% Ovalbumin for immunoassay.

EXAMPLE 2 Assay of Materials Provided by Control Methods and Method ofthe Invention

Test controls: To ensure that a standard approach was applied to alltests a 10 kg pool containing skeletal muscle and selected tissues foreach of bovine and ovine species were produced. Each was heated to 130°C. for 60 minutes and the resulting fluid decanted, cooled and passedthrough muslin to remove fat; further debris was removed bycentrifugation and the cleared supernatant retained. The processes ofgelatin extraction and protein concentration were applied to thesupernatant to produce protein pellets which were then stored at -70° C.and diluted as required for use.

Immunoassay: Two techniques were used: the microtechnique ofOuchterlony's double diffusion test, (Crowle (1973) Immunodifusion, 2ndEdit, Academic Press, New York 286-294) was used for simple screening ofantisera and to evaluate the protein extraction methods. Indirect ELISAusing horseradish peroxidase was used to allow amplification ofsensitivity and specificity to ruminant proteins, subject to thesuitability of the antisera.

Double Sandwich ELISA: Antibody conjugation: 20 mg of horseradishperoxidase (Merck Ltd) was activated with 1.0 ml of 0.1 M sodiumperiodate (Merck Ltd) and any unreacted reagent removed by gelfiltration using Sephadex G25 (Pharmacia). Activated peroxidase (HRPX)was conjugated with ruminant antibody after the method of Henning andNielsen (1987).

Plate coating: 50 μl of antibody were treated with 50 μl of glycinebuffer and diluted to a predetermined titre with Tris buffer asdescribed by Jackman (1992) Food Safety and Quality Assurance,application of immunoassay systems; M R A Morgan (Ed), Elsevier AppliedScience 215-226. 200 μl aliquots of this were then pipetted intomicrotitre wells. Each prepared plate was sealed and stood overnight at4° C. Non specific binding was prevented by washing plates with 0.01 Mphosphate buffered saline pH 7.2 containing 0.5% sucrose. 0.1%polyvinylpyrrolidone (PVP) and 0.05% Tween 20. After tapping dry plateswere dried at 37° C. for 16 to 24 hours sealed and stored if required at-70° C. for up to 3 months.

ELISA procedure: Optimal dilutions of coating antibody and HRPXconjugated antibody were determined by chequerboard titration againstcontrol extracts. Protein extracts were obtained from positive testcontrol material as described and from avian and porcine meat andbonemeal, used as negative controls, using the selection extractionmethod. All protein extracts were diluted as required using 2% ovalbuminwhich was also used as the reagent blank. 200 μl controls and reagentblank were added to duplicate wells of flat bottomed microtitre platestrips which had been precoated with coating antibody as previouslydescribed. The strips were sealed and incubated at 24° C. for 30 minutesthen washed in 0.05% Tween 20 in 0.1 M phosphate buffered salinesolution pH7.2. 200 μl of diluted HRPX antibody conjugate was added toeach well, the strips sealed and incubated at 24° C. for 30 minutes, thestrips washed as before and then 200 μl freshly prepared substrate,produced by mixing equal quantities of Chromagen E8073 and substratebuffer E8071 (Cambridge Veterinary Science) added to each well beforeshaking at 400 rpm for 6 minutes at 24° C. The reaction was stopped bythe addition of 50 μl of 1.8 M sulphuric acid to each well; theabsorbence value of each well being measured at 450 nm using anautomatic microtitre plate reader.

Evaluation of sera: All 15 sera were tested as both HRPX conjugates andprimary plate coating antisera giving a total of 225 test combinations.Each serum was used at a predetermined titre of 1/2000. For each HRPXconjugate/plate coating combination bovine and ovine controls at 1/200,avine and porcine meat and bonemeal extracts at 1/5 and reagent blank(2% ovalbumin in 0.1 M phosphate buffer) were tested. To permit anoverall comparison of results between each HRPX conjugate/plate coatingcombination a target optical density (OD) was set for the controlreadings and all other results extrapolated to meet the target OD. Fromthese results the HRPX conjugate/plate coating combinations for furtherdevelopment were selected using the following criteria: (i) low OD toavian/porcine meat and bonemeal extracts and the non-selected control;(ii) high ruminant detection titre and (iii) a low reagent backgroundreading. Three standard deviations above the mean reading of thenon-ruminant controls was allowed to offset the effect of anynon-specific background reading.

Results: Production of Antibody: The yield of antigen extracted from 1kg amounts of fresh muscle or tissue using a modification of the methodof Berger et al (1988) J. Association of Official Analytical Chemists7(2), 406-409 was considerably less than that obtained from the cookedmuscle tissue by the method of Kang'ethe and Gathuma (1987) MeatScience, 19,265-270. The yields, determined by protein assay using theSigma Micro-determination method, are shown in Table 2.

                  TABLE 2    ______________________________________    Extraction method for antigen    MATERIAL     Berger et al                           Kang'ethe & Gathuma    ______________________________________    Bovine muscle                 54.76     276.52    Bovine tissue                 50.11     234.52    Ovine tissue 39.07     195.73    Ovine tissue 136.54    442.13    Porcine muscle                 52.92     198.78    Porcine tissue                 129.00    301.10    Avian muscle 40.79     224.14    ______________________________________

When subject to isloelectric focusing using the Phast System at a pHgradient of 3.5-9.5 (Pharmacia UK Ltd) the main pI bands from the freshmuscle and tissue were found as follows: Identical pI bands were evidentat pH5.15 for bovine muscle and porcine tissue, pH5.5 for bovine tissueand ovine tissue and pH8.6 for bovine muscle and porcine muscle. Nomajor bands were found in the antigens extracted from cooked muscle ortissue.

Antisera production: Antisera specificity are given in Table 3 below.

                                      TABLE 3    __________________________________________________________________________    Ouchterlony's microtechnique double diffusion test results              Antigens (Fresh)  Antigens (Cooked)    Rabbit    Berger et al      Kang'ethe & Gathuma    No. Antiserum              BM BT                   OM OT                        PM PT                             AM BM BT                                     OM OT                                          PM PT                                               AM    __________________________________________________________________________    1   Bovine              3+ 4+                   -  - -  - -  4+ 4+                                     -  - -  - -        muscle (F)    2         3+ 4+                   +  - -  + -  4+ 4+                                     -  - -  - -    25        +  2+                   4+ 4+                        +  2+                             -  +  2+                                     2+ 2+                                          -  - -    9   Bovine              3+ 4+                   -  + -  - -  3+ 2+                                     +  - -  - -        tissue (F)    10        4+ 4+                   +  + -  + -  3+ 4+                                     +  - -  - -    3   Ovine +  + 4+ 2+                        -  + -  -  + +  2+                                          -  - -        muscle (F)    4         +  + 3+ 2+                        -  - -  -  + 2+ 2+                                          -  - -    11  Ovine 4+ 4+                   +  + -  - -  4+ 4+                                     -  - -  - -        tissue (F)    13  Bovine              +  2+                   -  + -  - -  3+ 3+                                     -  - -  - -        muscle (C)    14        4+ 4+                   +  + -  - -  3+ 4+                                     -  - -  - -    19  Bovine              +  + +  + -  - -  3+ 3+                                     -  + -  - -        tissue (C)    22        +  2+                   +  2+                        -  - -  4+ 3+                                     -  2+                                          -  - -    15  Ovine -  31                   2+ 2+                        -  - -  +  - 3+ 3+                                          -  - -        muscle (C)    16        +  + 2+ 2+                        -  - -  2+ + 3+ 3+                                          -  - -    23  Ovine 2+ + 2+ 4+                        +  + -  +  3+                                     3+ 4+                                          -  + -        tissue (C)    __________________________________________________________________________     + weak 2+ moderate 3+ strong 4+ very strong - negative     B bovine O ovine P porcine A avian M muscle T tissue F fresh C cooked

The majority of these antisera show a degree of cross-reactivity betweenbovine and ovine species antigens, with 5 out of 15 showing someresponse to porcine antigen.

The consistency and protein content of comparative gelatin solutions at4° C. following ammonium sulphate precipitation is shown in FIG. 1,while Table 4 demonstrates the effect of gelatin removal on the proteincontent of bovine and ovine meat and bonemeal.

                  TABLE 4    ______________________________________    Protein content of bovine and ovine meat and bonemeal    extract:                  Bovine    Ovine    ______________________________________    Total Protein   182 mg/ml   300 mg/ml    After gelatin and                    5.38 mg/ml  2.3 mg/ml    associated protein                    (2.95%)     (0.77%)    precipitation    Gelatin-free proteins                    0.8966 μg/ml                                0.3833 μg/ml    available in 1/6000    dilution of a sample    ______________________________________

Extraction methods:

The choice of extraction method was made by assessing the three methodsBerger et al (1988), Kang'ethe et al (1986) and Kang'ethe and Linqvist(1987); results being set out below in Table 5.

Evaluation of antisera:

Antisera were initially assessed against bovine and ovine meat andbonemeal extracts and the cross-reactivity found is set out in Table 6below.

                                      TABLE 5    __________________________________________________________________________    Comparison of protein extraction methods expressed as    maximum detectable protein dilution tested against pooled bovine    ovine antisera using Ouchterlony's microtechnique double    diffusion test.    Bovine extract dilution                          Ovine extract dilution    Method          0 2  4 8 16 32                        64                          0  2 4 8  16                                      32                                        64    __________________________________________________________________________    Gelatin          + +  + + +  + - +  + + +  + - -    removal    +protein    conc    Berger          - -  - - -  - - -  - - -  - - -    (1988)    Kangethe          + -  - - -  - - -  - - -  - - -    (1986)    Kangethe          + +  - - -  - - -  - - -  - - -    (1987)    __________________________________________________________________________

                  TABLE 6    ______________________________________    Detectable levels of antigen from gelatin free meat and    bonemeal extracts tested against each antigen using Ouchterlony's    microtechnique double diffusion test                   Maximum detectable dilution of    Rabbit Antiserum expressed                         meat and bonemeal protein extracts    No.    as immunogens used                         B       0     P     A    ______________________________________    1      B.M-F         1/32    1/16  0     0    2      "             1/64    1/2   Neat  0    25     "             1/4     1/32  0     0    9      B.T-F         1/32    1/16  0     0    10     "             1/32    1/16  0     0    3      0.M-F         1/16    1/16  0     0    4      "             1/16    1/32  Neat  0    11     0.T-F         1/64    1/16  0     0    13     B.M-C         1/16    0     0     0    14     "             1/16    0     0     0    19     B.T-C         1/32    1/8   0     0    22     "             1/16    1/16  0     0    15     0.M-C         1/4     1/8   0     0    16     "             1/4     1/16  0     0    23     0.T-C         1/16    1/32  0     0    ______________________________________

Indirect ELISA:

Direct comparison of the extrapolated results from the 225HRPXconjugate/plates coating antisera combinations revealed 38 givingacceptably high optical density (OD) readings to the bovine or ovinecontrols at 450 nm. Sixteen out of the 38 combinations of antisera/HRPXconjugates were selected on the basis of the 3 chosen criteria; thesebeing low OD of ≦0.08 for avian/porcine meat and bonemeal, high ruminantdetection titre of >1/2000 and a low reagent background OD reading of≦0.08.

Chequerboard titration: The optimum titre of sera of each of theselected HRPX conjugate/plate coating antisera combinations wasdetermined by running dilutions of both HRPX conjugate and plate coatingantisera against a fixed dilution of homologous control and meat andbonemeal extracts to produce the final assay protocol. It was found thatthe bovine and ovine systems could be separated on the basis of thechequerboard results.

Test evaluation:

Initially 50×20 g dry meat and bonemeal samples were tested doubleblind. Bovine, ovine, porcine and avian meat and bonemeals were mixed invarying proportions to simulate a range of ruminant contamination. Arepresentative sample could not be achieved below 1 part in 400 for a 20g dry mix because of the varied particulate composition of the meat andbonemeal; this was therefore a limiting factor for the dry mix test. Tenavian meat and bonemeal samples at a dilution of 1 part in 5 were usedas the non-ruminant controls and 3 standard deviations above the meanvalue chosen as the non-ruminant limit. All 50 test samples werecorrectly identified as ruminant or non-ruminant.

Results are shown in Table 7.

Sensitivity:

To evaluate the sensitivity of the test, liquid extracts of avian andporcine meat and bonemeals were contaminated with decreasing amounts ofliquid extracts of bovine and ovine meat and bonemeals. Both bovine andovine extracts were detectable up to the extinction dilution of 1/6400,but dilutions greater than this gave an optical density below the 3standard deviation non-ruminant control limit.

Results are shown in FIG. 2.

                  TABLE 7    ______________________________________    Double blind results on mixes of meat and bonemeal.    ______________________________________                                OD 450 nm    Sample  Mix composition     above NR    No.     A        P      B      0    control    ______________________________________    1        100     --      --     --  0    2       --       --     100    --   0.632    3       75       25     --     --   0    4       50       50     --     --   0    5       --       95     --     5    0.338    6       99.5     --     0.5    --   0.325    7       --       100    --     --   0    8       --       --     --     100  0.468    9       --       --     5      95   0.605    10      --       99.75  0.25   --   0.301    11      99.75    --     --     0.25 0.251    12      50       50     --     --   0    13      100      --     --     --   0    14      --       100    --     --   0    15      --       95     5      --   0.422    16      --       99.5   0.5    --   0.343    17      95       --     --     5    0.336    18      50       49.75  --     0.25 0.233    19      --       --     49.75  0.05 0.544    20      100      --     --     --   0    21      80       --     20     --   0.404    22      --       90     10     --   0.363    23      --       99     --     1    0.290    24      --       99     --     1    0.198    25      --       90     10     --   0.353    26      --       100    --     --   0    27      --       --     --     100  0.535    28      --       --     100    --   0.609    29      --       --     10     90   0.563    30      100      --     --     --   0    31      95       --     2.5    2.5  0.386    32      95       --     --     5    0.365    33      99       --     1      --   0.201    34      --       99.5   0.5    --   0.233    35      --       95     5      --   0.328    36      80       --     --     20   0.411    37      50       --     50     --   0.575    38      --       --     50     50   0.611    39      --       100    --     --   0    40      100      --     --     --   0    41      --       95     5      --   0.313    42      --       --     100    --   0.577    43      99       --     1      --   0.316    44      --       99.5   --     0.5  0.211    45      --       100    --     --   0    46      --       --     --     100  0.521    47      90       --     10     --   0.403    48      99.75    --     --     0.25 0.234    49      --       99.75  0.25   --   0.301    50      25       25     25     25   0.530    ______________________________________     NR = nonruminant

Evaluation of effect of temperature: The effect of temperature and timeupon the ruminant proteins and the efficacy of the immunoassay wereevaluated by heating small pieces of fresh bovine and ovine muscle atincreasing tempertures above 130° C. for up to 90 minutes. Extractstaken for each temperature were tested at 3 dilutions, ie. 1/1000,1/3200 and 1/6400, and the optical density plotted. The bovine musclewas detectable at all three dilutions at 155° C. but none weredetectable at 160° C. Ovine muscle detection declined rapidly and wasnot detectable at temperatures above 145° C. Results are shown in FIG.3. The sensitivity of the test to cooked ruminant proteins as comparedwith those produced by rendering, e.g. fish and bonemeal, issubstantially higher. The titres of detection therefore address onlythose of meat and bonemeal and meet the terms of this particularimmunoassay.

The results above show that the test method of the present inventionmakes it possible to detect ruminant heat stable proteins at 0.8966μg/ml for bovine species and 0.3833 μg/ml for ovine species.Furthermore, the cross-reactivity studies on antibodies show that whenraising antisera for use in the test method incorporating the samplepreparation method of the invention, these should be raisedheterologously to both fresh and cooked antigens, one each as thecoating antibody and signal generating antibody. Incorporation ofpolyvinylpyrrolidone (PVP) into the plate washing solution reducesbackground readings while further reduction occurs if 2% ovalbuminsolution is used to suspend the final concentrated sample. FIGS. 4a and4b illustrate the effects of these inclusions. Furthermore, for compoundfeeds it is found that use of the upper part of the range of ammoniumsulphate concentration, ie. about 70% by weight, e.g. 68%, is requiredto remove plant proteins and oils prior to protein concentration.

I claim:
 1. A method for pretreating heat stable proteins derivedmaterial from a rendered animal prior to performing an assay for theirpresence comprising the steps of:(a) preparing a protein-containingliquid extract from material from a rendered animal, (b) adding a saltto the extract to precipitate gelatin from the extract, (c) separatingthe gelatin precipitated from the extract, (d) increasing theconcentration of the salt until a portion of the remaining protein isprecipitated and isolating that protein.
 2. A method according to claim1 wherein in step (d), the remaining proteins are concentrated until ittests positive for proteins by immunoassay at a dilution of 1 in 6,000proteins extract to aqueous test medium when the proteins are derivedfrom a material obtained from rendered meat and bonemeal and at adilution of 1 in 400 when the proteins are derived from a material thathas been included in feeds.
 3. A method according to claim 1 whereinstep (a) comprises preparing a liquid buffered extract of the renderedmaterial and removing any solid material therefrom.
 4. A method asclaimed in claim 1 wherein the salt used is an ammonium salt.
 5. Amethod as claimed in claim 4 wherein the proteins are derived from amaterial which is of a compound feed and the salt is added to thebuffered extract to provide between a 30 and 70% weight salt solutionand wherein precipitation of gelatin and plant proteins occurs.
 6. Amethod for pretreating heat stable proteins derived from a renderedanimal material which has been incorporated into a compound feed, priorto performing an assay for their presence comprising preparing a liquidbuffered extract of the rendered material, removing solid material fromthe extract, adding an ammonium salt to the extract to provide from 30to 70% weight salt solution, separating the precipitate from theextract, increasing the concentration of the salt until a portion of theremainder of the proteins are precipitated, and isolating thoseproteins.
 7. A method as claimed in claim 6 wherein the proteins havenot been incorporated into a compound feed and sufficient of the salt isadded to the buffered extract to provide between a 30 and 40% weightsalt solution.
 8. A method as claimed in claim 6 wherein, in order toprecipitate the heat stable proteins after the first precipitate hasbeen removed, further salt is added to give a final concentration inexcess of 70% weight.
 9. A method as claimed in claim 8 wherein the saltis ammonium sulphate and is added to produce a final concentration inexcess of 90% weight.
 10. A method as claimed in claim 1 wherein theammonium sulphate is added to produce a final concentration of about92.5% weight.
 11. A method as claimed in claim 1 wherein the steps ofseparating gelatin and isolating heat stable proteins carried out usingcentrifugation.
 12. A method as claimed in claim 1 wherein the renderedanimal material is in the form of a mix of meat and bonemeal and thebuffer used to prepare the initial mix of meat and bonemeal confers aneutral pH.
 13. A method as claimed in claim 1 wherein the extract isprepared by blending the rendered animal material with the buffer usingan homogenizer, and then filtering the resultant mixture.